In biological microscopy, there has always been a continued drive towards increasing imaging resolution for relevant samples. In many cases, this drive translates to designing better optical systems to optimize for aberrations and resolution to achieve diffraction-limited performance. However, in cases where optical design has capped off and better resolution is still required, this drive leads to a need to extend the imaging resolution beyond the system's diffraction limit. Such a need has driven the development of many unique sub-diffraction imaging techniques that have made large impacts for microscopy.
These set of techniques can largely be divided into two classes. The first class of techniques is targeted towards situations where the sample is coherently illuminated and diffracts into the imaging system's aperture. In such cases, the general strategy to obtain sub-diffraction resolution makes use of the fact that imaging resolution is simply one of a few degrees of freedom that describe the imaging system. Though the total number of degrees of freedom is invariant, one can sacrifice the less relevant ones, such as temporal, polarization, or field-of-view constraints, to improve the final image resolution to beyond the conventional diffraction limit.
The second class of sub-diffraction resolution imaging techniques is a more recent development that has found great impact in biological fluorescence imaging. By appropriately utilizing properties of fluorophores, one can visualize a fluorescent sample at “super” resolutions beyond the diffraction limit. This class of “super-resolution” techniques is further subdivided into two main categories. The first main category is based on single molecule localization, where individual fluorescent emitters are localized at sub-diffraction resolution for each raw acquisition, and then aggregated into one final super-resolved image. Examples of such techniques include photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). The second category of super-resolution techniques uses spatially modulated excitation to narrow the effective imaging point-spread-function. Either this is done directly, as in stimulated emission depletion (STED) and ground-state depletion (GSD), or indirectly after post-processing, as in structured illumination microscopy (SIM). Out of these super-resolution techniques, SIM holds the unique advantage of potential extensions to non-fluorescent samples, and has shown exciting potential for such cases.
One difficulty with super-resolution techniques is that such techniques require fluorescent samples, and thus are ill suited for samples that are either not fluorescent or cannot be easily fluorescently tagged. To this end, synthetic aperture techniques allow sub-diffraction resolution imaging of non-fluorescent, diffractive samples by acquiring multiple electric-field maps of the sample taken at different illumination angles. Different regions of the sample's spatial frequency spectrum are covered by each illumination angle, and taken together, an effective optical passband larger than the system's physical one can be synthesized.
In view of the foregoing, there is a need for improved microscopy systems and techniques that extend the typical super-resolution concepts towards application in non-fluorescent imaging.